ExPEC glycoconjugate vaccine formulations

ABSTRACT

Compositions and methods for inducing an immune response against extra-intestinal pathogenic Escherichia coli (ExPEC) are described. In particular, multivalent vaccines containing E. coli antigen polysaccharide covalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein that can withstand multiple environmental stresses are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claim priority under 35 U.S.C. § 119 to Europeanapplication No. 16 195 256.9 filed on 24 Oct. 2016, the content of whichis hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions for inducing an immune responseagainst extra-intestinal pathogenic Escherichia coli (ExPEC). Inparticular, embodiments of this invention relate to multivalent vaccinescontaining conjugates of E. coli polysaccharide antigens covalentlybound to a carrier protein that can withstand multiple environmentalstresses.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “0281 US 00 ORD _ST25” and a creation date of Oct. 24, 2017,and having a size of 6,195 kB. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Extra-intestinal pathogenic E. coli (ExPEC) is the most commongram-negative pathogen in humans, and can cause various infectionsoutside of the gastrointestinal tract, which can lead to diverse andserious diseases, resulting in significant morbidity and mortality.Increasing multidrug resistance among ExPEC strains is an obstacle totreatment and leads to increasing numbers of hospitalizations and deathsand increasing healthcare costs associated with ExPEC infections.

A vaccine against ExPEC is therefore urgently needed. The O-antigen, acomponent of the surface lipopolysaccharide, has been identified as apromising vaccine target, and is used as antigen in a glycoconjugatevaccine that is currently under development (see, e.g. Poolman andWacker, 2016, J. Infect. Dis. 213: 6-13).

The glycoconjugate vaccines against ExPEC that are currently underdevelopment comprise 0-glycans of different serotypes of ExPEC, eachcoupled to a carrier protein, such as exoprotein A of Pseudomonasaeruginosa (EPA) (see e.g., WO2015/124769, and WO 2017/035181). Such avaccine comprising 0-glycans of the E. coli serotypes O25B, O1A, O2 andO6A is for instance in an ongoing phase 2 trial (ClinicalTrials.govIdentifier: NCT02546960).

It was found by the instant inventors that while the existing ExPECvaccine formulation (25 mM Tris pH 7.4, 2.7 mM KCl, 137 mM NaCl) isacceptable for short-term storage at 2-8° C. and in-use stability, it isnot robust upon freeze/thaw and under agitation stress. Accidentalfreezing, accidental heating, and agitation (e.g., during storage ortransportation) has a detrimental impact on product integrity of theExPEC formulations. For pharmaceutical products that are intended foruse in large populations, such as a glycoconjugate vaccine againstExPEC, it is beneficial to have a formulation that can be frozen in bulkat low temperatures, and after thawing can be stored at about 2-8° C.before usage (i.e., where the drug substance is frozen for large scaleand long term storage, but the drug product that is being used is storedat about 2-8° C., so at least one freeze-thaw cycle is inevitable forthe product). Further, the formulation would preferably also becompatible with different materials, such that the product (e.g., theglycoconjugate) can be stored in different formats (e.g., bags, bottles,vials, prefilled syringes, and/or applicable devices).

There is a need in the art for formulations of vaccines against ExPEC,that can withstand multiple environmental stresses (e.g., freeze/thaw,agitation, elevated temperature, light exposure, metal oxidant exposure,etc.) and result in a longer stability and longer shelf life of thecompositions, and preferably are compatible with multiple processing(e.g. container) materials. Any of the degradation routes resulting froman environmental stress can lead to lowered biological activity, and canpotentially also result in the formation of by-products or derivativesof the components of the formulations, thus resulting in increasedtoxicity and/or altered immunogenicity of the ExPEC vaccine. Therefore,a tailored approach is needed to find a robust formulation forglycoconjugate vaccines ensuring stability over a wide range ofconditions. Buffer type, pH, and specialized excipients will need to beselected, specifically combined, and subsequently meticulously optimizedto keep glycoconjugate vaccines chemically, physically, and biologicallystable. In view of all the factors that can vary, finding optimalconditions for formulating glycoconjugate vaccines against ExPEC isburdened with challenges, and the composition of a good formulation is apriori unpredictable.

Accordingly, there is a need in the art for formulations ofglycoconjugate vaccines against ExPEC that ensure that the vaccinecompositions can withstand multiple environmental stresses and have animproved stability and longer shelf life. It is the aim of the presentinvention to provide such formulations.

BRIEF SUMMARY OF THE INVENTION

Provided are two new formulations for glycoconjugate vaccines againstExPEC, providing the vaccine compositions with improved stabilizingeffect upon freeze/thaw, agitation stress, thermal stress, and metalinduced oxidation stress, which formulations are additionally compatiblewith various processing (e.g. container) materials. These improvedformulations can be utilized during storage or transportation of boththe drug product and the drug substance, where (accidental) freezing orharsh agitation can occur, which can have a detrimental impact onproduct integrity and therefore efficacy. Additionally, the novelformulations provided the glycoconjugate ExPEC vaccines with improvedstability against thermal stress, and therefore can have a broadcommercial application with respect to drug product and drug substancestorage, handling and transportation in a wide range of temperatures andconditions.

Provided herein are compositions comprising at least one E. coli Oantigen polysaccharide, wherein the at least one O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 3% to 8% (w/v) sorbitol; 5 to 15 mMmethionine; 5 to 20 mM potassium/sodium phosphate buffer at a pH of 6.5to 7.5; and 0.01% to 0.2% (w/v) surfactant. In preferred embodiments,the concentration of sorbitol is 4% to 6% (w/v). In preferredembodiments, the concentration of methionine is 8 to 12 mM. In preferredembodiments, the concentration of the potassium/sodium phosphate bufferis 8 to 15 mM. Preferably, the composition is a multivalent immunogeniccomposition comprising an E. coli O25B antigen polysaccharide, an E.coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide,and an E. coli O6A antigen polysaccharide, wherein each O antigenpolysaccharide is independently covalently bound to a exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein; 4% to 6% (w/v) sorbitol; 8to 12 mM methionine; 8 to 15 mM potassium/sodium phosphate buffer at apH of 6.5 to 7.5; and 0.01% to 0.2% (w/v) surfactant.

Also provided herein are compositions comprising at least one E. coli Oantigen polysaccharide, wherein the at least one O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 3% to 12% (w/v) sucrose; 0.1 to 1.5 mMEDTA; 5 to 20 mM potassium/sodium phosphate buffer at a pH of 6.5 to7.5; and 0.01% to 0.2% (w/v) surfactant. In preferred embodiments, theconcentration of sucrose is 3% to 10% (w/v). In preferred embodiments,the concentration of the potassium/sodium phosphate buffer is 8 to 15mM. Preferably, the composition is a multivalent immunogenic compositioncomprising an E. coli O25B antigen polysaccharide, an E. coli O1Aantigen polysaccharide, an E. coli O2 antigen polysaccharide, and an E.coli O6A antigen polysaccharide, wherein each antigen polysaccharide isindependently covalently bound to a exotoxin A of Pseudomonas aeruginosa(EPA) carrier protein; 3% to 10% (w/v) sucrose; 0.1 to 1.5 mM EDTA; 8 to15 mM potassium/sodium phosphate buffer at a pH of 6.5 to 7.5; and 0.01%to 0.2% (w/v) surfactant.

In certain embodiments, the E. coli O25B, O1A, O2, and O6A antigenpolysaccharides are at a weight ratio of 1:1:1:1 or 2:1:1:1.

In certain embodiments, the concentration of sorbitol is 5% (w/v).

In certain embodiments, the concentration of sucrose is 8% (w/v).

In certain embodiments, the concentration of methionine is 10 mM.

In certain embodiments, the concentration of EDTA is 1 mM.

In certain embodiments, the concentration of the potassium/sodiumphosphate buffer is 10 mM, and the pH of the potassium/sodium phosphatebuffer is 7.0.

In certain embodiments, the concentration of the surfactant is 0.02%(w/v). In preferred embodiments, the surfactant is composed of ahydrophilic head (an OH-group) and a long, hydrophobic tail(carbon-chain, which can comprise at least about 12, 14, 16, 18, 20, 30,40, 50, 60, 80, 100, 120, 130, or more carbons in the carbon chain). Inpreferred embodiments, the surfactant is a non-ionic surfactant. Incertain embodiments, the surfactant is selected from the groupconsisting of F-68, PS20, PS40, PS60, and PS80. In certain embodiments,the surfactant is PS80.

In certain embodiments, provided are compositions consisting essentiallyof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, wherein each O antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 5% (w/v) sorbitol; 10 mM methionine; 10 mM potassium/sodiumphosphate buffer at a pH of 7.0; and 0.02% PS80. Also provided arecompositions consisting of an E. coli O25B antigen polysaccharide, an E.coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide,and an E. coli O6A antigen polysaccharide, wherein each O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10 mM methionine;10 mM potassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80.

In certain embodiments, provided are compositions consisting essentiallyof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6Aantigen polysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 other E. coli O antigen polysaccharides,wherein each O antigen polysaccharide is covalently bound to a exotoxinA of Pseudomonas aeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10mM methionine; 10 mM potassium/sodium phosphate buffer at a pH of 7.0;and 0.02% PS80. Also provided are compositions consisting of an E. coliO25B antigen polysaccharide, an E. coli O1A antigen polysaccharide, anE. coli O2 antigen polysaccharide, an E. coli O6A antigenpolysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 other E. coli O antigen polysaccharides, whereineach O antigen polysaccharide is covalently bound to a exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10 mMmethionine; 10 mM potassium/sodium phosphate buffer at a pH of 7.0; and0.02% PS80.

In certain embodiments, provided are compositions consisting essentiallyof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6Aantigen polysaccharide, wherein each O antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphatebuffer at a pH of 7.0; and 0.02% PS80. Also provided are compositionsconsisting of an E. coli O25B antigen polysaccharide, an E. coli O1Aantigen polysaccharide, an E. coli O2 antigen polysaccharide, an E. coliO6A antigen polysaccharide, wherein each O antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphatebuffer at a pH of 7.0; and 0.02% PS80.

In certain embodiments, provided are compositions consisting essentiallyof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6Aantigen polysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 other E. coli O antigen polysaccharides,wherein each O antigen polysaccharide is covalently bound to a exotoxinA of Pseudomonas aeruginosa (EPA) carrier protein; 8% (w/v) sucrose; 1mM EDTA; 10 mM potassium/sodium phosphate buffer at a pH of 7.0; and0.02% PS80. Also provided are compositions consisting of an E. coli O25Bantigen polysaccharide, an E. coli O1A antigen polysaccharide, an E.coli O2 antigen polysaccharide, an E. coli O6A antigen polysaccharide,and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 other E. coli O antigen polysaccharides, wherein each O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 8% (w/v) sucrose; 1 mM EDTA; 10 mMpotassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80.

Also provided herein are methods of preparing compositions as disclosedherein. In certain embodiments, provided are methods of preparing acomposition comprising adding at least one E. coli O antigen covalentlylinked to an EPA carrier, water, salts for a buffer solution (i.e.sodium(di)hydrogen phosphate and potassium(di)hydrogen phosphate [i.e.Na₂HPO₄ and KH₂PO₄ or NaH₂PO₄ and K₂HPO₄]), a tonicity modifier (i.e.sorbitol or sucrose), an anti-oxidant (i.e. methionine if the tonicitymodifier is sorbitol; EDTA if the tonicity modifier is sucrose), and asurfactant (e.g. PS80) to a container, adjusting the pH to the desiredpH (i.e. 6.5 to 7.5, e.g. 7.0), and mixing these components such that aliquid formulation according to the invention is produced. In apreferred embodiment, methods of preparing the composition compriseadding an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, wherein each antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; water; potassium phosphate; sodium phosphate; sorbitol;methionine; and PS80 to a container, adjusting the pH to 6.5-7.5 (e.g.7.0), and mixing each component such that the final concentration of thepotassium/sodium phosphate buffer is 5-20 mM (e.g. 10 mM) with a pH of6.5-7.5 (e.g. 7.0), the final concentration of sorbitol is 3-8% (e.g.5%) (w/v), the final concentration of methionine is 5-15 mM (e.g. 10mM), and the final concentration of PS80 is 0.01-0.08% (e.g. 0.02%)(w/v). In another preferred embodiment, methods of preparing thecomposition comprise adding an E. coli O25B antigen polysaccharide, anE. coli O1A antigen polysaccharide, an E. coli O2 antigenpolysaccharide, an E. coli O6A antigen polysaccharide, wherein eachantigen polysaccharide is covalently bound to a exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein; water; potassiumphosphate; sodium phosphate; sucrose; EDTA; and PS80 to a container,mixing each component, and adjusting the pH to 6.5-7.5 (e.g. 7.0) suchthat the final concentration of sucrose is 3-12% (e.g. 8%) (w/v), thefinal concentration of EDTA is 0.1-1.5 mM (e.g. 1 mM), the finalconcentration of potassium/sodium phosphate buffer is 5-20 mM (e.g. 10mM) with a pH of 6.5-7.5 (e.g. 7.0), and the PS80 is 0.01-0.08% (e.g.0.02%) (w/v).

In certain embodiments, the compositions herein are provided as a liquidcomposition. By liquid composition, it is meant that the composition isin liquid form at 2-8° C., and preferably stored at 2-8° C.

In certain embodiments, the compositions can be stored and are stable at2-8° C., at 25° C., or at 40° C. In a preferred embodiment, thecomposition is stored and is stable at 2-8° C. In certain embodiments,the composition is stable at 2-8° C. for at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months. Incertain embodiments, the composition is stable at 25° C. for at leastabout 1, 2, 3, 4, 5, 6, or more months. In certain embodiments, thecomposition is stable at 40° C. for at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or more weeks.

In certain embodiments, the compositions herein are provided as a frozenformulation. By frozen formulation, it is meant that the composition isin a solid form when stored at or below about −18°, e.g. at about −20°C., −40° C., −60° C., −70° C., −80° C. or any temperature in between, orlower. In certain embodiments, the compositions can be stored and arestable at −40° C. or −60° C. depending on the tonicity modifier presentin the composition. In certain embodiments, the compositions can bestored and are stable at −70° C. In certain embodiments, the compositioncomprises sucrose and is stable at −40° C. for at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or more months. In certain embodiments,the composition comprises sorbitol and is stable at −60° C. for at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In certainembodiments the composition comprises sorbitol or sucrose and is stableat −70° C. for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreyears.

In preferred embodiments, the compositions of the invention do notcomprise sodium chloride.

In certain embodiments, the concentration for each O-antigenpolysaccharide in the composition is between about 1 and 200 μg/mL, e.g.between about 2 and 100 μg/mL, e.g. between about 4 and 50 μg/mL. Incertain embodiments thereof, the polysaccharide:carrier protein ratio isbetween about 1:10 and about 1:2, e.g between about 1:5 and 1:2 for eachO-antigen polysaccharide in the composition.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the present application, will be betterunderstood when read in conjunction with the appended drawings. Itshould be understood that the invention is not limited to the preciseembodiments shown in the drawings.

In the drawings:

FIGS. 1A, 1B and 1C show that PS20, PS80, and F68, but not sorbitol,were able to prevent freeze/thaw induced aggregation of the ExPECglycoconjugate under the test conditions: PS20, PS80, F68 and sorbitolwere added to an ExPEC glycoconjugate formulation and were subjected tofreeze/thaw induced stress,

FIG. 1A shows the integration scheme of a size exclusionchromatography-high performance liquid chromatography (SEC-HPLC)chromatogram;

FIG. 1B shows a SEC-HPLC chromatogram demonstrating the results of theexperiment before and after freeze/thaw; and

FIG. 1C shows a graph plotting the percentage values of the pre-peak 1upon total peak integration;

FIG. 2 shows a graph comparing percentage of SEC-HPLC chromatographicpre-peak 1 for formulations 26 and 28 head-to-head with an old ExPECglycoconjugate formulation, when subjected to 40° C. for an extendedtime period of 12 weeks: both upright (↑) and inverted (↓) vialorientations were examined, Formulations 26 and 28 demonstratedincreased stability over the old ExPEC glycoconjugate formulation whensubjected to the thermal stress (40° C.); and

FIGS. 3A, 3B and 3C show graphs demonstrating the pre-peak 1 trends forformulations 26 and 28 as compared to the old ExPEC glycoconjugateformulation when exposed to three different concentrations of tungstenextract (HW, MW, LW) and stored at 40° C. for 4 weeks.

FIG. 4 shows SEC-HPLC chromatograms demonstrating the results of theexperiment before and after agitation stress (TO is control, i.e. noagitation stress) for formulation 26 and 26 as compared to the oldformulation in contact with PETG and PC plastic materials.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentrationor a concentration range described herein, are to be understood as beingmodified in all instances by the term “about.” Thus, a numerical valuetypically includes ±10% of the recited value. For example, aconcentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, aconcentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).As used herein, the use of a numerical range expressly includes allpossible subranges, all individual numerical values within that range,including integers within such ranges and fractions of the values unlessthe context clearly indicates otherwise.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers can be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition.

It should also be understood that the terms “about,” “approximately,”“generally,” “substantially” and like terms, used herein when referringto a dimension or characteristic of a component of the preferredinvention, indicate that the described dimension/characteristic is not astrict boundary or parameter and does not exclude minor variationstherefrom that are functionally the same or similar, as would beunderstood by one having ordinary skill in the art. At a minimum, suchreferences that include a numerical parameter would include variationsthat, using mathematical and industrial principles accepted in the art(e.g., rounding, measurement or other systematic errors, manufacturingtolerances, etc.), would not vary the least significant digit.

As used herein, the terms “O polysaccharide”, “O-antigen”, “O antigen”,“O-antigen polysaccharide”, “O-polysaccharide antigen”, and theabbreviation “OPS”, all refer to the O antigen of Gram-negativebacteria, which is a component of the lipopolysaccharide (LPS) and isspecific for each serotype or sero(sub)type of the Gram-negativebacteria. The O antigen usually contains repeating units (RUs) of two toseven sugar residues. As used herein, the RU is set equal to thebiological repeat unit (BRU). The BRU describes the RU of an O-antigenas it is synthesized in vivo.

As used herein, the terms “conjugate” and “glycoconjugate” all refer toa conjugation product containing an E. coli O antigen covalently boundto a carrier protein (e.g., a exotoxin A of Pseudomonas aeruginosa(EPA)). The conjugate can be a bioconjugate, which is a conjugationproduct prepared in a host cell, wherein the host cell machineryproduces the O antigen and the carrier protein and links the O antigento the carrier protein, e.g., via N-links. The conjugate can also beprepared by other means, for example, by chemical linkage of the proteinand sugar antigen.

E. coli O antigens in embodiments of the invention include O1A, O2, O6A,and O25B, which are disclosed in WO2015/124769, and WO 2017/035181. Eachof these references is herein incorporated by reference in its entirety.Other E. coli O antigens can be used as well, e.g. in addition to thespecific O1A, O2, O6A and/or O25B antigens, and can for example include,but are not limited to, O antigens from E. coli O1, O2, O4, O6, O7, O8,O15, O16, O18, O21, O25, O73, O75 and O153 serotypes or subserotypesthereof.

As used herein, the term “drug substance” refers to the bulk product ofan individual glycoconjugate (E. coli O antigen polysaccharidecovalently coupled to EPA carrier protein, e.g. E. coli O25B O antigencovalently coupled to EPA), that is at higher concentration than theproduct as will be finally administered to a subject. The drug substancecan be produced after finalization of the downstream process to purifythe glycoconjugate. The drug substance can, for example, be stored in amore concentrated form in a formulation buffer of the invention, forinstance in frozen condition, e.g. at minus 70° C.

As used herein, the term “drug product” refers to the formulation of theglycoconjugates, in final form for administration to a subject. The drugproduct contains all the glycoconjugates (E. coli O antigenpolysaccharides coupled to EPA carrier protein in case of a multivalentvaccine, e.g. O25B, O1A, O2 and O6A, each individually coupled to an EPAcarrier protein, for a four-valent vaccine, and optionally more E. coliO antigens coupled to EPA carrier protein if the valency of the vaccineis increased). Drug product can typically be prepared by mixing the drugsubstances of the respective glycoconjugates, and dilution byformulation buffer if needed, such that the target dose of vaccine isproduced. Drug product in a formulation according to the invention canbe stored at 2-8° C., and remains stable at that temperature for atleast 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months.

The term “about,” when used in conjunction with a number, refers to anynumber within ±1, ±5 or ±10% of the referenced number.

When the O antigen is covalently bound to a protein carrier, theeffective amount or dosage for the O antigen is calculated based on onlythe O antigen polysaccharide moiety in the conjugate.

Diseases associated with ExPEC or ExPEC infections include, but are notlimited to, urinary tract infection, surgical-site infection,bacteremia, abdominal or pelvic infection, pneumonia, nosocomialpneumonia, osteomyelitis, cellulitis, pyelonephritis, wound infection,meningitis, neonatal meningitis, peritonitis, cholangitis, soft-tissueinfections, pyomyositis, septic arthritis, and sepsis.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy. The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Forexample, a first therapy (e.g., a composition described herein) can beadministered prior to, concomitantly with, or subsequent to theadministration of a second therapy (e.g., a composition describedherein, or another therapy, e.g., treatment with an antibiotic) to asubject.

As used herein, the term “subject” refers to an animal, preferably amammal, and may include a non-primate (e.g., a pig, horse, goat, sheep,cat, dog, rabbit, rat, or mouse) and a primate (e.g., a monkey,chimpanzee, or a human). In certain embodiments, a subject is anon-human animal. In another embodiment, a subject is a human. The terms“subject” and “patient” can be used herein interchangeably. Thecompositions according to the invention are suitable for administrationto a subject, and can be used to generate an immune response againstExPEC O-antigens that are encompassed in the composition.

As used herein, an “immunological response” or “immune response” to anantigen or composition refers to the development in a subject of ahumoral and/or a cellular immune response to the antigen or an antigenpresent in the composition.

Compositions Comprising E. coli O Antigen Polysaccharides

In one general aspect, the invention relates to a multivalent vaccinecontaining O-antigen serotypes found predominantly among E. coliclinical isolates, which can be used to provide active immunization forthe prevention of disease caused by ExPEC having the O-antigen serotypescontained in the vaccine. In one embodiment, the invention relates to acomposition comprising at least one E. coli O antigen polysaccharide. Incertain embodiments, the composition can comprise an E. coli O25Bantigen polysaccharide. In other embodiments, the composition comprisesan E. coli O1A, O2, and/or O6A antigen polysaccharide. In preferredembodiments, the composition comprises E. coli O1A, O2, O6A and O25Bantigen polysaccharides, which are disclosed in WO2015/124769, and WO2017/035181. Each of these references is herein incorporated byreference in its entirety. In certain embodiments, other or additionalE. coli O antigen polysaccharide is present in the composition. Such E.coli O antigen polysaccharides can include, but are not limited to, Oantigens from (sub)serotypes of E. coli O1, O2, O4, O6, O7, O8, O15,O16, O18, O21, O25, O73, O75 and O153. Depending on the need, thecomposition can include more than one additional E. coli O antigens,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more, additional E. coli O antigens, to provide immune protectionagainst multiple E. coli serotypes.

In certain embodiments, the compositions and methods can relate to theE. coli O25B antigen and one or more additional E. coli O antigens.Examples of E. coli O antigens can include, but are not limited to E.coli O25B, O1A, O2 and O6A antigens.

As used herein an “E. coli O25B antigen” refers to an O antigen specificto the E. coli O25B serotype. In one embodiment, an E. coli O25B antigencomprises the structure of Formula O25B′:

wherein the n in Formula O25B′ is an integer of 1 to 30, 1 to 20, 1 to15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment ofthe invention, the n in Formula O25B′ is an integer of 10-20.

As used herein, an “E. coli O1A antigen” refers to an O antigen specificto the E. coli O1A serotype. In one embodiment, an E. coli O1A antigencomprises the structure of Formula O1A′:

wherein then in Formula O1A′ is an integer of 1 to 30, 1 to 20, 1 to 15,1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, thenin Formula O1A′ is an integer of 7-15.

As used herein, an “E. coli O2 antigen” refers to an O antigen specificto the E. coli O2 serotype. In one embodiment, an E. coli O2 antigencomprises the structure of Formula O2′:

wherein the n in Formula O2′ is an integer of 1 to 30, 1 to 20, 1 to 15,1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, thenin Formula O2′ is an integer of 8-16.

As used herein, an “E. coli O6 antigen” refers to an O antigen specificto the E. coli O6 serotype. In one embodiment, an E. coli O6 antigen isan E. coli O6A.

As used herein, an “E. coli O6A antigen,” also referred to as “E. coliO6K2 antigen” or “E. coli O6Glc antigen,” refers to an O antigenspecific to the E. coli O6A serotype. In one embodiment, an E. coli O6Aantigen comprises the structure of Formula O6A′:

wherein the β 1, 2 linkage is also named β2 linkage, the n in FormulaO6A′ is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25,10 to 20, or 15 to 20. In one embodiment, the n in Formula O6A′ is aninteger of 8-18.

The E. coli O antigen polysaccharides in the compositions of theinvention are covalently bound (sometimes referred to as “conjugated”)to a carrier protein, the carrier protein of the invention beingExotoxin A of P. aeruginosa, preferably a detoxified variant thereof(EPA; see, e.g., Ihssen, et al., (2010) Microbial cell factories 9, 61;WO2015/124769). The combination of carrier protein and O antigenpolysaccharide is referred to as glycoconjugate. Typically, O antigenfrom each E. coli serotype in the composition is covalently bound to aseparate carrier protein, i.e. if the composition comprises four Oantigen polysaccharides, each of these is separately and independentlycoupled to the EPA carrier protein, and the composition thus comprisesfour different glycoconjugates. One way of making the glycoconjugates isby bioconjugation, wherein the host cell machinery produces the Oantigen and the carrier protein and links the O antigen to the carrierprotein, e.g., via N-links (see e.g. WO2015/124769 for the preparationof bioconjugates of E. coli O antigen O25B, O1A, O2 and O6Apolysaccharides to EPA carrier protein). For EPA, various detoxifiedprotein variants have been described in literature and could be used ascarrier proteins. A detoxified (or non-toxic) EPA refers to anyPseudomonas aeruginosa exotoxin A that lacks ADP ribosylation activity.The ribosylation activity of the wild type EPA is located between aboutamino acids 400 and 613 of EPA, and for example deleting amino acidGlutamic acid at position 553 from a wild-type EPA, or substitutingHistidine at position 426 with Tyrosine in a wild-type EPA, detoxify theEPA molecule. Other amino acids within amino acids 400-613 of wild-typeEPA can be modified by, e.g. deletion, addition, or substitution ofamino acid residues to eliminate ADP ribosylation activity and therewithtoxicity, as known to the skilled person. In preferred embodiments ofthe invention, the EPA carrier protein is a detoxified variant. Incertain non-limiting and exemplary embodiments, the EPA carrier proteincan comprise an amino acid sequence as set forth in SEQ ID NO: 1 (beingan embodiment of a detoxified EPA), or an amino acid sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:1, or has 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids difference to SEQ ID NO: 1, anyof these optionally further comprising 1, 2, 3, 4, 5, 6, 7, 8, or morerecombinantly introduced glycosylation consensus sequences as describedbelow. In specific embodiments, introduction of glycosylation sites isaccomplished by insertion of glycosylation consensus sequences (e.g.,Asn-X-Ser(Thr), wherein X can be any amino acid except Pro; orpreferably Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independentlyselected from any natural amino acid except Pro (SEQ ID NO: 2) (see,e.g., WO 2006/119987 and WO2015/124769)) anywhere in the primarystructure of the EPA protein. In preferred embodiments, the EPA carrierprotein comprises one or more recombinantly introduced N-linkedglycosylation consensus sites having amino acid sequenceAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO: 2). In certainembodiments, the EPA carrier protein comprises one, two, three, four,five, six, seven, eight, or more of such introduced consensus sequences.In other embodiments, the EPA does not comprise such introduced N-linkedglycosylation consensus sites, for instance when the glycoconjugate isprepared using classical conjugation chemistry (see e.g., U.S. Pat. No.5,370,872; Cryz et al, 1990, Infection and Immunity 58: 373-377).

In certain embodiments, the E. coli O-antigens are covalently bound tothe carrier protein at a polysaccharide-to-protein weight/weight ratioof about 1:20 to 20:1, preferably 1:10 to 10:1, e.g. 1:10 to 3:1. Incertain non-limiting embodiments for bioconjugates, the ratio ofpolysaccharide/protein is between about 0.1 and 0.5 (i.e.polysaccharide:protein is about 1:10 to 1:2, e.g. about 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value in between) for eachbioconjugate, depending on the O-antigen serotype.

In certain non-limiting embodiments, the E. coli O antigenpolysaccharides can be present in the composition at a concentration ofabout 1 to 200 μg/mL for each O antigen polysaccharide, e.g., about 2 to100 μg/mL, e.g. about 4 to 50 μg/mL, e.g. about 4 to 48 μg/mL, e.g.about 8 to 48 μg/mL, e.g. about 4 to 32 μg/mL, e.g. about 8 to 32 μg/mL,e.g., 8, 16, 20, or 32 μg/mL or any value in between. Preferably, in acomposition of at least two E. coli O antigen polysaccharides, each Oantigen polysaccharide is present in weight ratios of between 1:1 to1:2, or any value in between, for each combination of O antigenpolysaccharides in the composition.

In certain non-limiting embodiments the O-antigen polysaccharides arepresent in the composition at a concentration of total O-antigenpolysaccharides of about 4 to 1000 μg/mL, e.g. about 10 to 500 μg/mL,e.g. about 20 to 250 μg/mL, e.g. about 24 to 120 μg/mL.

In certain non-limiting embodiments, the total concentration for the EPAcarrier protein in the composition can for instance be between about 40and 2000 μg/mL, e.g. about 100 to 1500 μg/mL, e.g. about 200 to 1200μg/mL, e.g. about 250 to 600 μg/mL.

In certain embodiments, the composition comprises O25B, O1A, O2 and O6Aantigen polysaccharides at a weight ratio of 1:1:1:1, wherein each ofthe O antigen polysaccharides is covalently bound to an EPA carrierprotein. In another embodiment, the composition comprises the E. coliO25B, O1A, O2 and O6A antigen polysaccharides at a weight ratio of2:1:1:1, wherein each of the O antigen polysaccharides is covalentlybound to an EPA carrier protein. In each of such embodiments, the O25Bantigen polysaccharide can, as a non-limiting example, be present at aconcentration of about 8, 16, or 32 μg/mL.

The compositions described herein are useful in the treatment andprevention of infection of subjects (e.g., human subjects) with ExPEC.In certain embodiments, in addition to comprising E. coli O antigenpolysaccharides covalently bound to an EPA carrier protein, thecompositions described herein comprise a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier,” as used herein in the context of a pharmaceuticallyacceptable carrier, refers to a diluent, adjuvant, excipient, or vehiclewith which the pharmaceutical composition is administered. Water in thecompositions of the invention is preferably water for injection.

The compositions provided herein comprise a surfactant. Surfactants asused herein are organic compounds that are amphiphilic, meaning theycontain both hydrophobic groups (their tails) and hydrophilic groups(their heads). In preferred embodiments, the surfactant is composed of ahydrophilic head (comprising an OH-group) and a long hydrophobic tail(carbon-chain, which can comprise at least about 12, 14, 16, 18, 20, 30,40, 50, 60, 80, 100, 120, 130, or more carbons in the carbon chain).Preferred surfactants according to the invention are non-ionicsurfactants. Examples of surfactants suitable for compositions of theinvention include, but are not limited to, polysorbate 20 (PS20),polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 80 (PS80), andPluronic® F-68 (F-68). In a preferred embodiment, the surfactant isPS80. In certain embodiments, the surfactant in the composition isprovided at a concentration of 0.01% (weight/volume (w/v)) to 0.2%(w/v). In certain embodiments, the surfactant in the composition isprovided at a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%,0.17%, 0.18%, 0.19%, 0.2%, or any value in between. For embodimentswhere F-68 is the surfactant, the concentration is preferably from about0.05% to about 0.2%. For embodiments where PS20, PS40, PS60, or PS80 isthe surfactant, the concentration is preferably from about 0.01% toabout 0.08%, e.g. about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, or any value in between.

The compositions provided herein further comprise a buffer with aspecific pH. According to the present invention, the compositioncomprises a potassium/sodium phosphate buffer. Concentration ranges forsuch buffer can, for example, be about 5 mM to about 20 mM, about 8 mMto about 15 mM, about 8 mM to about 13 mM, about 9 mM to about 11 mM. Incertain embodiments, the buffer is provided at a concentration of 8 mM,9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or any value in between.In certain embodiments, the buffer concentration is from about 8 mM toabout 15 mM. In a preferred embodiment, the composition comprises thebuffer at a concentration of 10 mM. pH ranges for such buffers can, forexample, be about pH 6.5 to about pH 7.5, about pH 6.6 to about pH 7.4,about pH 6.7 to about pH 7.3, about pH 6.8 to about pH 7.2, about pH 6.9to about pH 7.1. In certain embodiments, the pH can be 6.5, 6.6, 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or any value in between. In apreferred embodiment the pH of the buffer is 7.0. In a preferredembodiment, the composition comprises a potassium/sodium phosphatebuffer at a concentration of 10 mM and at a pH of 7.0.

Potassium/sodium phosphate buffer solutions can, for example, comprisepotassium from K₂HPO₄ (dibasic) or KH₂PO₄ (monobasic) and sodium fromNa₂HPO₄ (dibasic) or NaH₂PO₄ (monobasic). In certain embodiments, thepotassium/sodium phosphate buffer comprises a monobasic potassiumphosphate (KH₂PO₄) and a dibasic sodium phosphate (Na₂HPO₄). In certainembodiments, the potassium/sodium phosphate buffer comprises a dibasicpotassium phosphate (K₂HPO₄) and a monobasic sodium phosphate (NaH₂PO₄).The molar ratio between the monobasic phosphate species and the dibasicphosphate species ([H₂PO₄]⁻/[HPO₄]²⁻), regardless of the counter ion,ranges between 5.13 and 0.52.

The compositions provided herein can further comprise a tonicitymodifier (sometimes also referred to as ‘stabilizer’). The tonicitymodifiers used in the composition of the invention are either sucrose orsorbitol, depending on the other excipients, as defined herein. Incertain embodiments, the composition comprises sucrose. In certainembodiments, the composition comprises sucrose at a concentration rangeof about 3% to about 12% (w/v), about 3% to about 11% (w/v), about 3% toabout 10% (w/v), about 4% to about 10% (w/v), about 5% to about 9%(w/v), about 6% to about 9% (w/v). In certain embodiments, the sucroseis provided at a concentration of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, or any value in between. In a preferred embodiment, the compositioncomprises sucrose at a concentration of about 3% to about 10%. In apreferred embodiment, the composition comprises sucrose at aconcentration of 8%. In other preferred embodiments, the compositioncomprises sorbitol. In a preferred embodiment, the composition comprisessorbitol at a concentration of about 3% to about 8% (w/v), about 3% toabout 7% (w/v), about 4% to about 6% (w/v). In preferred embodiments,the composition comprises sorbitol at a concentration of about 4% toabout 6% (w/v). In certain embodiments, the composition comprisessorbitol at a concentration of about 3%, 4%, 5%, 6%, 7%, 8%, or anyvalue in between. In a preferred embodiment, the composition comprisessorbitol at a concentration of 5%.

The compositions provided herein further comprise an anti-oxidant. Theanti-oxidant used in the compositions of the present invention is EDTAor methionine, depending on the other excipients, as described herein.In certain embodiments, the composition comprises EDTA. In certainembodiments, the composition comprises EDTA at a concentration of about0.1 mM to about 1.5 mM, about 0.2 mM to about 1.4 mM, about 0.5 mM toabout 1.3 mM, about 0.7 mM to about 1.2 mM, about 0.8 mM to about 1.2mM, about 0.9 mM to about 1.1 mM. In certain embodiments, the EDTA isprovided at a concentration of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM,0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM,1.5 mM, or any value in between. In a preferred embodiment, thecomposition comprises EDTA at a concentration of 1.0 mM. Thecompositions of the invention that comprise EDTA also comprise sucrose.In other embodiments, the composition comprises methionine. In certainembodiments, the composition comprises methionine at a concentration ofabout 5 mM to about 15 mM, about 6 mM to about 14 mM, about 7 mM toabout 13 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM. In apreferred embodiment, the composition comprises methionine at aconcentration of about 8 mM to about 12 mM. In certain embodiments, themethionine is provided at a concentration of 5 mM, 6 mM, 7 mM, 8 mM, 9mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or any value in between.In a preferred embodiment, the composition comprises methionine at aconcentration of 10 mM. The compositions of the invention that comprisemethionine also comprise sorbitol.

Provided are compositions comprising an E. coli O25B antigenpolysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide, and an E. coli O6A antigen polysaccharide,wherein each O antigen polysaccharide is covalently bound to a exotoxinA of Pseudomonas aeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10mM methionine; 10 mM potassium/sodium phosphate buffer at a pH of 7.0;and 0.02% PS80. Also provided are compositions consisting essentially ofan E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, wherein each O antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 5% (w/v) sorbitol; 10 mM methionine; 10 mM potassium/sodiumphosphate buffer at a pH of 7.0; and 0.02% PS80. Also provided arecompositions consisting of an E. coli O25B antigen polysaccharide, an E.coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide,and an E. coli O6A antigen polysaccharide, wherein each O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10 mM methionine;10 mM potassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80.Also provided are compositions comprising an E. coli O25B antigenpolysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide, an E. coli O6A antigen polysaccharide, and 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 otherE. coli O antigen polysaccharides, wherein each O antigen polysaccharideis covalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA)carrier protein; 5% (w/v) sorbitol; 10 mM methionine; 10 mMpotassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80. Alsoprovided are compositions consisting essentially of an E. coli O25Bantigen polysaccharide, an E. coli O1A antigen polysaccharide, an E.coli O2 antigen polysaccharide, an E. coli O6A antigen polysaccharide,and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 other E. coli O antigen polysaccharides, wherein each O antigenpolysaccharide is covalently bound to a exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; 5% (w/v) sorbitol; 10 mM methionine;10 mM potassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80.Also provided are compositions consisting of an E. coli O25B antigenpolysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide, an E. coli O6A antigen polysaccharide, and 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 otherE. coli O antigen polysaccharides, wherein each O antigen polysaccharideis covalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA)carrier protein; 5% (w/v) sorbitol; 10 mM methionine; 10 mMpotassium/sodium phosphate buffer at a pH of 7.0; and 0.02% PS80.

Provided are compositions comprising an E. coli O25B antigenpolysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide, an E. coli O6A antigen polysaccharide, whereineach O antigen polysaccharide is covalently bound to a exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein; 8% (w/v) sucrose; 1 mMEDTA; 10 mM potassium/sodium phosphate buffer at a pH of 7.0; and 0.02%PS80. Also, provided are compositions consisting essentially of an E.coli O25B antigen polysaccharide, an E. coli O1A antigen polysaccharide,an E. coli O2 antigen polysaccharide, an E. coli O6A antigenpolysaccharide, wherein each O antigen polysaccharide is covalentlybound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein;8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer ata pH of 7.0; and 0.02% PS80. Also provided are compositions consistingof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6Aantigen polysaccharide, wherein each O antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphatebuffer at a pH of 7.0; and 0.02% PS80. Also provided are compositionscomprising an E. coli O25B antigen polysaccharide, an E. coli O1Aantigen polysaccharide, an E. coli O2 antigen polysaccharide, an E. coliO6A antigen polysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O antigenpolysaccharides, wherein each O antigen polysaccharide is covalentlybound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein;8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer ata pH of 7.0; and 0.02% PS80. Also provided are compositions consistingessentially of an E. coli O25B antigen polysaccharide, an E. coli O1Aantigen polysaccharide, an E. coli O2 antigen polysaccharide, an E. coliO6A antigen polysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O antigenpolysaccharides, wherein each O antigen polysaccharide is covalentlybound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein;8% (w/v) sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer ata pH of 7.0; and 0.02% PS80. Also provided are compositions consistingof an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6Aantigen polysaccharide, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 other E. coli O antigen polysaccharides,wherein each O antigen polysaccharide is covalently bound to a exotoxinA of Pseudomonas aeruginosa (EPA) carrier protein; 8% (w/v) sucrose; 1mM EDTA; 10 mM potassium/sodium phosphate buffer at a pH of 7.0; and0.02% PS80.

The compositions described herein can optionally additionally comprise apreservative, such as the mercury derivative thimerosal, phenoxyethanol,or parabens. In a specific embodiment, the pharmaceutical compositionsdescribed herein comprise 0.001% to 0.01% thimerosal. In otherembodiments, the pharmaceutical compositions described herein do notcomprise a preservative.

In certain embodiments, the compositions described herein (e.g., theimmunogenic compositions) comprise, or are administered in combinationwith, an adjuvant. The adjuvant for administration in combination with acomposition described herein can be administered before, concomitantlywith, or after administration of said composition. In some embodiments,the term “adjuvant” refers to a compound that when administered inconjunction with or as part of a composition described herein augments,enhances and/or boosts the immune response to a bioconjugate, but whenthe adjuvant compound is administered alone does not generate an immuneresponse to the bioconjugate. Adjuvants can enhance an immune responseby several mechanisms including, e.g., lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages. Incertain embodiments, the compositions described herein do not comprisean adjuvant besides the bioconjugates and excipients, and/or are notadministered in combination with an adjuvant besides the bioconjugatesand the excipients (in case the bioconjugates or excipients wouldcomprise some intrinsic adjuvant properties, these would be disregardedand no additional extrinsic adjuvant would be added in theseembodiments).

Specific examples of adjuvants include, but are not limited to, aluminumsalts (alum) (such as aluminum hydroxide, aluminum phosphate, andaluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (seeUnited Kingdom Patent GB2220211), MF59 (Novartis), AS03(GlaxoSmithKline), AS04 (GlaxoSmithKline), imidazopyridine compounds(see WO2007/109812), imidazoquinoxaline compounds (see WO2007/109813)and saponins, such as QS21 (see Kensil et al., in Vaccine Design: TheSubunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY,1995); U.S. Pat. No. 5,057,540). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., 1997, N. Engl. J. Med. 336, 86-91). Another adjuvant is CpG(Bioworld Today, Nov. 15, 1998).

In certain embodiments, the compositions described herein are formulatedto be suitable for the intended route of administration to a subject.For example, the compositions described herein can for instance beformulated to be suitable for intramuscular, subcutaneous, parenteral,oral, intranasal, intradermal, transdermal, colorectal, intraperitoneal,intratracheal, topical, rectal or pulmonary administration. In certainembodiments, the compositions described herein are useful foradministration by intramuscular injection. In other embodiments, thecompositions described herein can be administered intradermally. Inother embodiments, the compositions described herein can be deliveredvia the skin.

In another aspect, also provided herein are drug substance compositionscomprising at least one of the four conjugates described in detailherein (i.e. representing O-antigen from E. coli serotypes O25B, O1A,O2, or O6A), in the formulations described herein. Such compositionsthat comprise only one or a subset of the four conjugates can forinstance be useful for storage of bulk antigens, e.g. as drug substancebefore compounding into the final drug product, and share usefulstability characteristics described for the drug product compositionscomprising all of the four conjugates.

Methods/Uses

The compositions of the invention can, for instance, be used for amethod of inducing an immune response to ExPEC in a subject in needthereof. Preferably, the immune response is effective to prevent ortreat a disease associated with ExPEC in the subject in need thereof.The method comprises administering to the subject a compositionaccording to the invention.

In certain embodiments, compositions provided herein can be stored in acontainer. Suitable containers can include, but are not limited to,bags, vials, syringes, bottles, and test tubes. In certain embodiments,a vial with a stopper capable of being pierced by a syringe comprisesany of the compositions described herein. The containers provided hereincan be formed from a variety of materials such as glass (e.g.,borosilicate glass), metal, or plastic (e.g., polycarbonates). Incertain embodiments, the container is for instance a vial of type Iborosilicate glass. In other embodiments, the container is a glasssyringe with either luer lock or staked needle. In other embodiments,the container is a vial or syringe of plastic material, such aspolycarbonate (PC) or polyethylene terephthalate glycol (PETG), whichmaterial for instance was shown to be compatible with drug substanceaccording to the invention. Vials may optionally contain a rubberstopper, for instance from (chloro/bromo)butyl rubber coated with afluoropolymer film (e.g. Flurotec [ethylene tetrafluoroethylene (EFTE)]or Teflon [fluorinated ethylene propylene (FEP)]). Other materials andcontainer types can also be used, and compatibility with theformulations of the invention can be determined by those skilled in theart based upon the present disclosure.

In certain embodiments, drug substance is stored in polycarbonatecontainers, e.g. bottles. In certain embodiments, drug substance isstored in polyethylene terephthalate glycol containers, e.g. bottles. Incertain embodiments, drug product is stored in glass containers, e.g.vials.

The formulations provided herein improve the stability of theglycoconjugates in the compositions. By stable, it is generally meantthat the composition retains its physical stability and/or chemicalstability and/or biological activity upon storage. Preferably, thecomposition essentially retains its physical and chemical stability andits biological activity upon storage. Physical stability, chemicalstability, and biological activity can be determined by those of skillin the art utilizing methods disclosed herein and methods known in theart. For the present invention, a composition is considered ‘stable’(and the corresponding formulation is considered ‘stabilizing’), ifthere is 5% or less change in the size-exclusion chromatography HPLCpre-peak (indicative of aggregation of the glycoconjugate) as comparedto time point zero, as described in the examples herein. For example,the existing ExPEC vaccine composition (in 25 mM Tris pH 7.4, 2.7 mMKCl, 137 mM NaCl) shows a more than 5% increase of the pre-peak at 8weeks at 40° C., and is thus not stable at 8 weeks at this temperature,whereas the compositions of the invention show less than 5% increase ofthe pre-peak after 12 weeks at 40° C. and are thus stable for at least12 weeks at this temperature. The compositions of the invention arestable in a glass container at 2-8° C. for at least 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, or more, months. The compositions of theinvention are also stable in plastic containers for at least 7 days at25° C., which allows processing of the ExPEC glycoconjugate productunder these conditions.

In certain embodiments, the compositions herein are provided as a liquidcomposition. By liquid composition, it is meant that the composition isin liquid form when at 2-8° C., and preferably stored at 2-8° C.Optionally, the liquid composition is stored at 25° C. or at 40° C., forinstance for accelerated stability testing under thermal stressconditions.

In certain embodiments, the compositions can be stored and are stable at2-8° C., at 25° C., or at 40° C. In a preferred embodiment, thecomposition is stored and is stable at 2-8° C. In certain embodiments,the composition is stable at 2-8° C. for at least about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, or more, months. In certain embodiments,the composition is stable at 25° C. for 1, 2, 3, 4, 5, 6, or moremonths. In certain embodiments, the composition is stable at 40° C. forat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks.

In certain embodiments, the compositions herein are provided as a frozencomposition. By frozen composition, it is meant that the composition isin a solid form when stored at or below about −18°, e.g. at about −20°C., −40° C., −60° C., −70° C., −80° C. or any temperature in between, orlower. In certain embodiments, the compositions can be stored and arestable at −40° C. or −60° C. depending on the tonicity modifier presentin the composition. In certain embodiments, the compositions can bestored and are stable at −70° C. In certain embodiments, the compositioncomprises sucrose and is stable at −40° C. for at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or more months. In certain embodiments,the composition comprises sorbitol and is stable at −60° C. for at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In certainembodiments the composition comprises sorbitol or sucrose and is stableat −70° C. for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreyears. The compositions of the present invention are designed to be morestable than the old composition formulation described previously.Stability of the composition can be determined by methods describedherein and methods known in the art. Utilizing these methods, thecompositions of the present invention will be more stable over a giventime period at a given temperature as compared to compositions of theold formulation over the same time period and same temperature.

Also provided herein are methods of preparing compositions as disclosedherein. In certain embodiments, provided are methods of preparing acomposition comprising adding at least one E. coli O antigen covalentlylinked to an EPA carrier, water, salts for a buffer solution (i.e.sodium(di)hydrogen phosphate and potassium(di)hydrogen phosphate [i.e.Na₂HPO₄ and KH₂PO₄ or NaH₂PO₄ and K₂HPO₄]), a tonicity modifier (i.e.sorbitol or sucrose), an anti-oxidant (i.e. methionine if tonicitymodifier is sorbitol, EDTA if tonicity modifier is sucrose), and asurfactant (e.g. PS80) to a container, adjusting the pH to the desiredpH (i.e. 6.5 to 7.5, e.g. 7.0), and mixing these components such that aliquid formulation according to the invention is produced. In apreferred embodiment, methods of preparing the composition compriseadding an E. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, wherein each antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; water; potassium phosphate; sodium phosphate; sorbitol;methionine; and PS80 to a container, adjusting the pH to 7.0, and mixingeach component such that the final concentration of the potassium/sodiumphosphate buffer is 10 mM with a pH of 7.0, sorbitol is 5% (w/v),methionine is 10 mM, and PS80 is 0.02% (w/v). One, non-limiting andexemplary, way of preparing a formulation according to the invention isas follows: to about 3.5 liter of water is added: 3.3696 g KH₂PO₄(Mw=136.09 g/mol), 2.1635 g Na₂HPO₄ (Mw=141.96 g/mol), 200 g sorbitol,5.9684 g Methionine (Mw=149.21 g/mol), 8 mL of 10% (w/v) PS80 stock,which results in a pH of about 6.73, which is subsequently adjusted withabout 5004 of 10 N NaOH to target pH=7.0, and the volume is adjusted to4 liter with water. A formulation comprising sucrose and EDTA instead ofsorbitol and methionine can be prepared in an analogous manner, which isclear to the skilled person having common general knowledge and theinformation provided herein. One possible way to prepare a compositionaccording to the invention is by buffer exchange, e.g. using tangentialflow filtration, filtration, dialysis, size exclusion chromatography, orthe like, to exchange the formulation that is prepared as describedabove for any buffer in which the ExPEC glycoconjugate is present, forinstance during or after a purification step of the ExPECglycoconjugate. Such a buffer exchange step is routine for a skilledperson, using the information provided herein.

The following examples of the invention are to further illustrate thenature of the invention. It should be understood that the followingexamples do not limit the invention and that the scope of the inventionis to be determined by the appended claims.

EMBODIMENTS

Embodiment 1 is an immunogenic composition comprising one or more of anE. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, preferably all four of the E. coli O25B, O1A, O2and O6A antigen polysaccharides, wherein each antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 3% to 8% (preferably 4% to 6%) (w/v) sorbitol; 5 to 15 mM(preferably 8 to 12 mM) methionine; 5 to 20 mM (preferably 8 to 15 mM)potassium/sodium phosphate buffer at a pH of 6.5 to 7.5; and 0.01% to0.2% (w/v) surfactant.

Embodiment 2 is the immunogenic composition of embodiment 1, wherein theE. coli O25B, O1A, O2, and O6A antigen polysaccharides are at a weightratio of 1:1:1:1 or 2:1:1:1.

Embodiment 3 is the immunogenic composition of embodiment 1 or 2,wherein the concentration of sorbitol is 5% (w/v).

Embodiment 4 is the immunogenic composition of any of embodiments 1-3,wherein the concentration of methionine is 10 mM.

Embodiment 5 is the immunogenic composition of any of embodiments 1-4,wherein the concentration of the potassium/sodium phosphate buffer is 10mM, and the pH of the potassium/sodium phosphate buffer is 7.0.

Embodiment 6 is the immunogenic composition of any of embodiments 1-5,wherein the surfactant comprises a hydrophilic head and a hydrophobictail, preferably, wherein the surfactant is selected from the groupconsisting of F-68, PS20, and PS80.

Embodiment 7 is the immunogenic composition of embodiment 6, wherein thesurfactant is PS80.

Embodiment 8 is the immunogenic composition of embodiment 7, wherein theconcentration of the surfactant is 0.02% (w/v).

Embodiment 9 is an immunogenic composition comprising:

-   -   a. an E. coli O25B antigen polysaccharide, an E. coli O1A        antigen polysaccharide, an E. coli O2 antigen polysaccharide,        and an E. coli O6A antigen polysaccharide, wherein each antigen        polysaccharide is covalently bound to a exotoxin A of        Pseudomonas aeruginosa (EPA) carrier protein;    -   b. 5% (w/v) sorbitol;    -   c. 10 mM methionine;    -   d. 6.19 mM KH₂PO₄ and 3.81 mM Na₂HPO₄ buffer at a pH of 7.0; and    -   e. 0.02% (w/v) PS80.

Embodiment 10 is an immunogenic composition comprising one or more of anE. coli O25B antigen polysaccharide, an E. coli O1A antigenpolysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6Aantigen polysaccharide, preferably all four of the E. coli O25B, O1A, O2and O6A antigen polysaccharides, wherein each antigen polysaccharide iscovalently bound to a exotoxin A of Pseudomonas aeruginosa (EPA) carrierprotein; 3% to 12% (preferably 3 to 10%) (w/v) sucrose; 0.1 to 1.5 mMEDTA; 5 to 20 mM (preferably 8 to 15 mM) potassium/sodium phosphatebuffer at a pH of 6.5 to 7.5; and 0.01% to 0.2% (w/v) surfactant.

Embodiment 11 is the immunogenic composition of embodiment 10, whereinthe E. coli O25B, O1A, O2, and O6A antigen polysaccharides are at aweight ratio of 1:1:1:1 or 2:1:1:1.

Embodiment 12 is the immunogenic composition of embodiment 10 or 11,wherein the concentration of sucrose is 8% (w/v).

Embodiment 13 is the immunogenic composition of any of embodiments10-12, wherein the concentration of EDTA is 1 mM.

Embodiment 14 is the immunogenic composition of any of embodiments10-13, wherein the concentration of the potassium/sodium phosphatebuffer is 10 mM, and the pH of the potassium/sodium phosphate buffer is7.0.

Embodiment 15 is the immunogenic composition of any of embodiments10-14, wherein the surfactant comprises a hydrophilic head and ahydrophobic tail, preferably wherein the surfactant is selected from thegroup consisting of F-68, PS20, and PS80.

Embodiment 16 is the immunogenic composition of embodiment 15, whereinthe surfactant is PS80.

Embodiment 17 is the immunogenic composition of embodiment 16, whereinthe concentration of the surfactant is 0.02% (w/v).

Embodiment 18 is an immunogenic composition comprising:

-   -   a. an E. coli O25B antigen polysaccharide, an E. coli O1A        antigen polysaccharide, an E. coli O2 antigen polysaccharide,        an E. coli O6A antigen polysaccharide, wherein each antigen        polysaccharide is covalently bound to a exotoxin A of        Pseudomonas aeruginosa (EPA) carrier protein;    -   b. 8% (w/v) sucrose;    -   c. 1 mM EDTA;    -   d. 10 mM potassium/sodium phosphate (e.g. 6.19 mM KH₂PO₄ and        3.81 mM Na₂HPO₄) buffer at a pH of 7.0; and    -   e. 0.02% (w/v) PS80.

Embodiment 19 is the immunogenic composition of any one of embodiments1-18, wherein the concentration for each O-antigen polysaccharide isbetween about 1 and 200 μg/mL, preferably between 1 and 100 μg/mL, morepreferably between 2 and 50 μg/mL, e.g. between about 4 μg/mL and 32μg/mL.

Embodiment 20 is the immunogenic composition of any one of embodiments1-19, wherein the polysaccharide:carrier protein (weight:weight) ratiois between about 1:10 and about 1:2, e.g. between about 1:5 and about1:2 for each O-antigen polysaccharide.

Embodiment 21 is the immunogenic composition of any one of embodiments1-20 in a liquid form suitable for administration by injection orinfusion.

Embodiment 22 is the immunogenic composition of any one of embodiments1-21 for inducing an immune response in a subject in need thereof.

Embodiment 23 is use of the immunogenic composition of any one ofembodiments 1-21 for the manufacture of a medicament for inducing animmune response in a subject in need thereof.

Embodiment 24 is a composition according to any one of embodiments 1-21in a glass container.

Embodiment 25 is a composition according to any one of embodiments 1-21in a polycarbonate container.

Embodiment 26 is a composition according to anyone of embodiments 1-21in a polyethylene terephthalate glycol container.

Embodiment 27 is a composition according to any one of embodiments 1-21in a vial.

Embodiment 28 is a composition according to any one of embodiments 1-21in a syringe.

Embodiment 29 is a composition according to any one of embodiments 1-21or 24-28, which is stable for at least 6 months, preferably at least 12months, more preferably at least 18 months, more preferably at least 24months, more preferably at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, or more, months, when stored at a temperature of 2-8° C.

Embodiment 30 is a method to stably maintain a liquid immunogeniccomposition comprising an E. coli O antigen covalently coupled to an EPAcarrier protein, comprising storing a composition according to any oneof embodiments 1-21 or 24-29 at a temperature of 2-8° C. for at least 6months, preferably at least 12 months, more preferably at least 18months, more preferably at least 24 months, more preferably at least 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or more, months.

EXAMPLES Example 1: Addition of Surfactant Prevents Freeze/Thaw InducedAggregation of ExPEC Glycoconjugate

To determine which combination of excipients could be added to the E.coli O25B, O1A, O2, O6A antigen polysaccharides each independentlycovalently bound to a separate (i.e. total four separateglycoconjugates) exotoxin A of Pseudomonas aeruginosa (EPA), hereinafterreferred to as the ExPEC glycoconjugate, to aid with stabilization uponfreeze/thaw, agitation, thermal induced stress, and metal-inducedoxidation stress, different excipients were added to an initial ExPECglycoconjugate formulation (ExPEC glycoconjugate, 25 mM Tris, pH 7.4,137 mM NaCl, 2.7 mM KCl) (hereinafter referred to as the “Old”formulation, which is the formulation currently used in a phase 2clinical trial for the ExPEC glycoconjugate, ClinicalTrials.govIdentifier: NCT02546960), and the resulting formulation was tested forincreased stabilization by the appropriate methods.

In the old formulation (sometimes also referred to as “control” herein),the ExPEC glycoconjugate aggregated upon being subjected to freeze/thawinduced stress. Aggregation of the ExPEC glycoconjugate is visible as apre-peak in a size exclusion chromatography-high performance liquidchromatography (SEC-HPLC) profile (see, e.g., FIGS. 1A and 1B), in whichthe pre-peak 1 is an indicator for instability upon being subjected to astress.

Initially, a cryoprotectant (e.g., sorbitol) was added to the ExPECglycoconjugate formulation and the subsequent formulation was tested todetermine if the stability was increased upon being subjected tofreeze/thaw induced stress. It was found that the addition of sorbitoldid not protect the ExPEC glycoconjugate from freeze/thaw inducedaggregation as measured by SEC-HPLC (FIG. 1B).

Surprisingly however, addition of a surfactant (e.g., F-68 [also knownas poloxamer 188, or Pluronic F-68], PS20, or PS80) to the ExPECglycoconjugate formulation increased the stability of the ExPECglycoconjugate when subjected to freeze/thaw induced stress.Specifically, the surfactant prevented aggregation of the ExPECglycoconjugate when exposed to a freeze/thaw induced stress. Addition of0.01% PS-80, 0.01% PS 20, or 0.1% F-68 (all w/v) to the ExPECglycoconjugate prevented freeze/thaw induced aggregation as measured bySEC-HPLC (FIG. 1B and FIG. 1C). Each tested surfactant (all non-ionicsurfactants) was composed of a hydrophilic head (comprising an OH-group)and a long hydrophobic tail (carbon-chain, which can comprise at leastabout 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 120, 130, or morecarbons in the carbon chain). While not wishing to be bound by thetheory, it is believed that these physical properties allowed thesurfactants tested to prevent freeze/thaw induced aggregation of theExPEC glycoconjugate. In further development, PS80 was used as theexcipient to prevent freeze/thaw induced aggregation, as this particularsurfactant has additional advantages related to easy implementation forlate stage development of the ExPEC glycoconjugate.

Example 2: Formulation Evaluation for Buffer, pH Value, and TonicityModifier

After demonstrating that surfactants are capable of preventingfreeze/thaw induced aggregation of the ExPEC glycoconjugate, a suitablebuffer, pH, and a tonicity modifier for the formulation wasinvestigated. In a follow-up experiment, several formulations wereinvestigated, covering different buffer-pH combinations, in the presenceof NaCl (150 mM) or 5% sorbitol (w/v) as a tonicity modifier, and allcontaining PS80 (0.01% (w/v). Old formulation (25 mM Tris, pH 7.4, 137mM NaCl, 2.7 mM KCl) was used as a control. Multiple concentrations ofthe ExPEC glycoconjugate (e.g. 16 or 8 μg/mL for each polysaccharide)were tested, but we did not observe an effect of concentrationdifferences in stabilization of the formulations tested (data notshown).

Several stresses were applied to these formulations includingfreeze/thaw, agitation, light exposure, and thermal stress at varioustemperatures (e.g., 2-8° C., 25° C., and 40° C.) (Table 1).

TABLE 1 Stress conditions applied to reformulated ExPEC glycoconjugateformulations Stress Conditions Time Points Temperature 5° C. 0, 4, 8, 12weeks 25° C. 2, 4, 8 weeks 40° C. 1, 2, 4 weeks Agitation Vortex (1000rpm at 4 hours ambient temp) Freeze/Thaw −70° C. to 5 consecutive cyclesambient temp Photosensitivity Light exposure 200 W/m2 UV-A light (ICHQ1B option 2) (~6 hours) + 1200 klux-hr visible light (~40 hours)

TABLE 2 Analytical package for the analysis of reformulated ExPECglycoconjugate formulations Analytical Method Purpose Visual InspectionAppearance and clarity pH Acidity or basicity of samples OsmolalityOsmolality SEC-HPLC Purity, aggregate, cleavages Reverse phase (RP)-HPLCPurity, chemical modifications Isoelectric Focusing (IEF) Purity,chemical modifications Dynamic light scattering (DLS) Sub-visibleparticles FlowCAM Sub-visible particles

Utilizing the analytics package described in Table 2, it wasdemonstrated that formulations containing histidine at pH 7 or potassiumphosphate at pH 7 were more stable compared to other buffer-pHcombinations (data not shown). Also, at extreme pH values (i.e., pH 5.0and pH 8.0), there appears to be more aggregation of the ExPECglycoconjugate. Additionally, it was observed that formulationscontaining sorbitol as a tonicity modifier were unexpectedly morestabilizing than formulations containing NaCl as the tonicity modifier.In fact, it was observed that formulations containing NaCl were not ableto stabilize the ExPEC glycoconjugate against aggregation during storageat 40° C. When sorbitol was used as a tonicity modifier, aggregation ofExPEC glycoconjugate did not occur, demonstrating that sorbitol has asurprisingly stabilizing effect. Further, the aggregation at the pHextremes (i.e., pH 5.0 and pH 8.0) is reduced in sorbitol containingformulations compared to the NaCl containing formulations. This showsthat sorbitol makes the formulation more robust and capable of providinga stabilizing effect for the ExPEC glycoconjugate even if pH differencesoccur during production.

Example 3: Formulation Evaluation for Stability

Further formulations were designed to identify the best candidateformulations with respect to stability of the ExPEC glycoconjugate. Forthe buffers and pH, histidine was evaluated at a pH of 6.5 and 7.0, andinstead of purely potassium phosphate, a potassium/sodium phosphatebuffer (KH₂PO₄/Na₂HPO₄) was evaluated at a pH of 6.5 and 7.0. Thecombination of potassium and sodium was selected based upon initialresults, and better covers the local pH buffering capacity uponfreeze/thaw. Potassium dominates over sodium in the preparation of thebuffering system (e.g., for a 10 mM phosphate buffer with pH=7.0, weused 6.19 mM KH₂PO₄ and 3.81 mM Na₂HPO₄). As tonicity modifiers, sucrose(8% (w/v)) and sorbitol (5% (w/v)) were evaluated. Additionally, effectsof addition of anti-oxidant EDTA (1 mM) or methionine (10 mM) wasevaluated. It was also tested if absence of NaCl or presence of sorbitolprovided the protective effect observed in example 2, by including aformulation which comprised a combination of these (each at half theconcentration as compared to when these components were usedindividually as in example 2). All formulations contained PS80 at aconcentration of 0.02% (w/v).

The different formulations were subjected the same stress conditionsdescribed in example 2. Based on the combination of the stability data,a selection step was conducted, where the performance of eachformulation was evaluated and excluded based on the following criteria:(a) an additional post-peak was visible on RP-HPLC chromatogram in atleast two out of three time points for each temperature; (b) thepre-peak 1 was visible/increased on a SEC-HPLC chromatogram in at leasttwo out of three time points for each temperature; (c) an additionalpost-peak was visible on a SEC-HPLC chromatogram after subjected to astress (e.g., agitation, freeze/thaw, light exposure); and (d) thepre-peak 1 was visible/increased on a SEC-HPLC after subjected to astress (e.g., agitation, freeze/thaw, light exposure). It was found thattwo particular formulations, named formulations 26 and 28 herein, werecapable of providing the ExPEC glycoconjugate to withstand each stresscondition tested.

Formulation 26 comprised 10 mM Na/K phosphate buffer pH 7.0, 5% (w/v)sorbitol, 10 mM methionine, 0.02% (w/v) PS-80, and the ExPECglycoconjugate.

Formulation 28 comprised 10 mM Na/K phosphate buffer pH 7.0, 8% sucrose,1 mM EDTA, 0.02% PS-80, and the ExPEC glycoconjugate.

Surprisingly, the specific combinations of the tonicity modifier andanti-oxidant were relevant, as formulations comprising either (i)sorbitol with methionine, or (ii) sucrose with EDTA, outperformed (a)formulations wherein sorbitol was combined with EDTA, as well as (b)formulations wherein sucrose was combined with methionine, and (c)formulations wherein no anti-oxidant was present. Also surprisingly, itwas found that ExPEC glycoconjugate formulations preferably do notcontain sodium chloride. The formulations 26 and 28 can be varied withinranges, while still being expected to stabilize the ExPECglycoconjugate. Table 3 provides the applicable ranges for pH andexcipient concentrations for formulations 26 and 28.

TABLE 3 Ranges for pH and excipient concentrations for ExPECglycoconjugate formulations Excipient Range pH 6.5 7.5 K/Na Phosphate(mM) 5 20 Sorbitol (%) (w/v) 3 8 Sucrose (%) (w/v) 3 12 Methionine (mM)5 15 EDTA (mM) 0.1 1.5 Surfactant (%): F-68 0.05 0.2 PS20 0.01 0.08 PS800.01 0.08

Formulations 26 and 28 were further tested in a confirmation study tocompare head-to-head with the old ExPEC glycoconjugate formulation(ExPEC glycoconjugate, 25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl).Both upright and inverted vial orientations were tested to assess theimpact of the contact with the stopper of the final container.Formulations 26 and 28 are composed of excipients that do not pose anysafety concerns at the proposed concentrations, are used in licensedvaccines and/or are listed as approved excipients for parenteraladministration. The combination of these excipients for formulations 26and 28 and the pH value of the buffering solution contribute to theimproved stabilizing effect of the ExPEC glycoconjugate compared to theold ExPEC glycoconjugate formulation. The proposed combination of theseexcipients for formulations 26 and 28 demonstrated to preserve the drugsubstances (DSs) and the drug product (DP) of the ExPEC glycoconjugatevaccine upon freeze/thaw and thermal stress (e.g., FIG. 2), whilecomplying with the expected stability trending upon storage at 2-8° C.and 25° C. (data not shown). Polysaccharide concentration tested for theDP was 20 μg/mL for each strain (80 μg/mL in total), and proteinconcentration was 300 μg/mL in total. For a DS of serotype O25B (i.e.containing an E. coli O25B antigen polysaccharide covalently bound toEPA carrier protein), the tested concentration of polysaccharide was 200μg/mL and the tested concentration of protein was 830 μg/mL. Suchcompositions were stable over time. So far, the drug product informulations 26 and 28 showed stability for at least 6 months at 2-8°C., at least six months at 25° C., and stability studies are ongoing.

Example 4: Formulation Evaluation for Metal Induced Oxidation Stress

Formulations 26 and 28 were further evaluated for their stabilizingeffect for the ExPEC glycoconjugate in the presence of tungsten, as amodel for metal-induced product oxidation. Tungsten residues are usuallypresent in the tip of glass pre-filled syringes (PFS), which is theresult of the tip forming process with a tungsten pin. The deposition oftungsten residues in the PFS depends on the manufacturing process andcan vary between PFS manufacturers in the range of 250-1250nanograms/barrel. Tungsten has been associated with protein aggregationin PFSs (Jiang et al., J. Pharmaceutical Sci. 98(12): 4695-710 (2009);Seidl et al., Pharmaceutical Res. 29:1454-67 (2012)). Thus, thestability of formulations 26 and 28 and the old formulation wereinvestigated when exposed to tungsten at different concentrations whilemonitoring oxidative stress and propensity of aggregation of the ExPECglycoconjugate. In this evaluation, three levels of tungsten (high (HW),medium (MW), and low (LW)) were examined covering all the levels oftungsten residue present in PFS available currently in the market. Thestress conditions applied to the formulations are summarized in Table 4.

TABLE 4 Stress conditions applied to tungsten containing formulationsStress Conditions Time Points Temperature 40° C. 0, 1, 2, and 4 weeksAgitation Vortex (200 rpm at 24 hours ambient temp)

Formulations 26 and 28 were shown to have the least amount of aggregatesof the ExPEC glycoconjugate over time, as demonstrated by SEC-HPLCanalysis (stability-indicating pre-peak 1). Pre-peak levels at 4 weeksonly reached 3.1% as compared to 1.0% at T-0, even when exposed to thehighest levels of tungsten (averaging 0.5% growth per week, FIGS. 3A, 3Band 3C).

The invention thus provides two different improved liquid formulationsfor ExPEC glycoconjugate vaccine. The first, most preferred, compositioncomprises the ExPEC glycoconjugate, sorbitol, methionine, K/Na-phosphatebuffer pH 7 and surfactant. The second preferred composition comprisesthe ExPEC glycoconjugate, sucrose, EDTA, K/Na-phosphate buffer pH 7 andsurfactant. These compositions have the advantage over the previouslydescribed composition that upon storage at 2-8° C., the ExPECglycoconjugate vaccine shows a better stability profile in the newformulations of the invention, where aggregation and degradation productformation is prevented, as shown by stability indicating assays(SEC-HPLC and RP-HPLC). The stabilizing effect of both new inventedformulations is even more evident upon freeze/thaw, thermal (40° C.),and metal induced oxidation stress when analyzed with the aforementionedanalytical techniques. These features allow multiple storage andtransportation options for both DS and DP for ExPEC glycoconjugatevaccine. In addition, the improved properties with regards towithstanding metal-induced oxidation stress, could also be applied inlong term storage of the ExPEC vaccine in alternative primary packagingsystems (e.g. prefilled syringe and/or applicable devices).

Example 5: Formulation Evaluation for Compatibility with PlasticMaterials

Formulations 26 and 28 were further evaluated for their stabilizingeffect for the ExPEC glycoconjugate drug substance (O25B conjugate,tested concentration of polysaccharide 227-242 μg/mL and protein952-1048 μg/mL) in contact with various plastic materials such aspolycarbonate (PC) and polyethylene terephthalate glycol (PETG). Plasticmaterials have been associated with protein degradation, e.g. when theplastic material had been sterilized by irradiation. The formulationswere tested for stability under harsh agitation stress (24 hours at 200rpm at room temperature) in PETG and PC containers. The old formulationdemonstrated complete degradation under these conditions in the PETGcontainer. In contrast, as demonstrated by SEC-HPLC analysis (see FIG.4, using pre-peak 1 as an indicator for instability), formulations 26and 28 remained stable under the same conditions in the tested PETG andPC containers. In addition, stability of the drug substance in theseformulations stored for seven days in PC and PETG containers at 25° C.was observed (data not shown).

What is claimed is:
 1. An immunogenic composition comprising: a. an E.coli 025B antigen polysaccharide, an E. coli O1A antigen polysaccharide,an E. coli 02 antigen polysaccharide, and an E. coli O6A antigenpolysaccharide, wherein each antigen polysaccharide is independentlycovalently bound to an exotoxin A of Pseudomonas aeruginosa (EPA)carrier protein; b. 3% to 8% (w/v) sorbitol; c. 5 to 15 mM methionine;d. 5 to 20 mM phosphate buffer comprising potassium phosphate and sodiumphosphate at a pH of 6.5 to 7.5; and e. 0.01% to 0.2% (w/v) surfactant.2. The immunogenic composition of claim 1, wherein the concentration ofsorbitol is 5% (w/v).
 3. The immunogenic composition of claim 1, whereinthe concentration of methionine is 10 mM.
 4. The immunogenic compositionof claim 1, wherein the concentration of the phosphate buffer is 10 mM,and the pH of the phosphate buffer is 7.0.
 5. The immunogeniccomposition of claim 1, wherein the surfactant is a non-ionicsurfactant.
 6. The immunogenic composition of claim 5, wherein thesurfactant is polysorbate 80 (PS80).
 7. An immunogenic compositionconsisting of: a. an E. coli 025B antigen polysaccharide, an E. coli O1Aantigen polysaccharide, an E. coli 02 antigen polysaccharide, and an E.coli O6A antigen polysaccharide, wherein each antigen polysaccharide isindependently covalently bound to an exotoxin A of Pseudomonasaeruginosa (EPA) carrier protein; b. 5% (w/v) sorbitol; c. 10 mMmethionine; d. 10 mM phosphate buffer comprising KH₂PO₄ and Na₂HPO₄ at apH of 7.0; e. 0.02% (w/v) polysorbate 80 (PS80); and f. water.
 8. Theimmunogenic composition of claim 1, which is stable for at least 6months when stored at a temperature of 2-8° C.
 9. The immunogeniccomposition of claim 1, wherein the concentration for each 0-antigenpolysaccharide is about 1 to 200 μg/mL.
 10. A method for stablymaintaining a liquid immunogenic composition comprising an E. coliO-antigen covalently coupled to an EPA carrier protein, the methodcomprising storing a composition according to claim 1 at a temperatureof 2-8° C. for at least 6 months.
 11. The immunogenic composition ofclaim 7, wherein the concentration for each 0-antigen polysaccharide isabout 1 to 200 μg/mL, and the immunogenic composition is stable for atleast 6 months when stored at a temperature of 2-8° C.
 12. Theimmunogenic composition of claim 1, wherein the surfactant is selectedfrom the group consisting of poloxamer 188, polysorbate 20 (PS20), andpolysorbate 80 (PS801.
 13. The immunogenic composition of claim 6,wherein the concentration of PS80 is 0.02% (w/v).
 14. A method forstably maintaining a liquid immunogenic composition comprising an E.coli O-antigen covalently coupled to an EPA carrier protein, the methodcomprising storing a composition according to claim 7 at a temperatureof 2-8° C. for at least 6 months.
 15. An immunogenic compositioncomprising: a. an E. coli 025B antigen polysaccharide, wherein theantigen polysaccharide is covalently bound to an exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein; b. 3% to 8% (w/v)sorbitol; c. 5 to 15 mM methionine; d. 5 to 20 mM phosphate buffercomprising potassium phosphate and sodium phosphate at a pH of 6.5 to7.5; and e. 0.01% to 0.2% (w/v) surfactant, wherein the immunogeniccomposition is stable for at least 6 months when stored at a temperatureof 2-8° C.
 16. The immunogenic composition of claim 15, wherein theconcentration of sorbitol is 5% (w/v).
 17. The immunogenic compositionof claim 15, wherein the concentration of methionine is 10 mM.
 18. Theimmunogenic composition of claim 15, wherein the concentration of thephosphate buffer is 10 mM, and the pH of the phosphate buffer is 7.0.19. The immunogenic composition of claim 15, wherein the surfactant is anon-ionic surfactant selected from the group consisting of poloxamer188, polysorbate 20 (PS20), and polysorbate 80 (PS80).
 20. Theimmunogenic composition of claim 15, wherein the surfactant ispolysorbate 80 (PS80) and the concentration of PS80 is 0.02% (w/v).